Linker peptide for constructing fusion protein

ABSTRACT

A linker peptide for constructing a fusion protein. The linker peptide comprises a flexible peptide and a rigid peptide. The flexible peptide consists of one or more flexible units. The rigid peptide consists of one or more rigid units. The flexible unit comprises two or more amino acid residues selected from Gly, Ser, Ala, and Thr. The rigid unit comprises a human chorionic gonadotropin β-subunit carboxy-terminal peptide (CTP) bearing a plurality of glycosylation sites. The linker peptide can more effectively eliminate mutual steric hindrance of two fusion molecules, decreasing a reduction/loss of polymerization or activity resulting from improper folding of an active protein or a conformational change. On the other hand, the negatively charged, highly sialylated CTP can resist renal clearance, further prolonging a half-life of a fused molecule and enhancing bioavailability of a fused protein. Furthermore, a protective effect of a glycosylated side chain CTP can lower the protease sensitivity of the linker peptide, making a linker region of the fusion protein less degradable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national stage entry under 35U.S.C. § 371 of International Application No. PCT/CN2016/106011, filedon Nov. 16, 2016 and published as WO 2018/032638 A1, which claimspriority to Chinese Patent Application Nos. 201610692679.4, filed onAug. 19, 2016, and 201610694914.1, filed on Aug. 19, 2016. The contentsof these applications are each incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format. Said ASCII copy, created onNov. 20, 2020, is named 15079_0002-00000_SL.txt and is 49,929 bytes insize.

Please insert the sequence listing, filed herewith in electronic format,into the application before the claims.

FIELD OF THE INVENTION

The present invention relates to the field of fusion proteins and, morespecifically, to a peptide linker for the construction of fusionproteins.

BACKGROUND

In recent two decades, protein fusion technology has been widely used inthe construction of bifunctional antibodies, bifunctional enzymes, andbifunctional proteins. However, a variety of problems have beenencountered in the construction of fusion proteins. For example,proteins that fold correctly during expression alone do not foldproperly in the fusion protein; the active site is blocked after fusiondue to the short distance between the two fused proteins; the fusionprotein molecule is easily degraded by proteases when it cannot foldproperly or when its conformation has changed; the protein catalyticdomain with certain flexibility loses its original function afterfusion; and so on. The emergence of these problems often leads toreduction or even complete loss of the activity of the fusion proteins.It is generally believed that the activity of the original proteinmolecule will decrease to a certain extent after the protein molecule isconstructed in the fusion protein. A favorable fusion protein is the onethat keeps more than 50% activity of the original protein molecule(s).In order to solve the above problems, researchers conducted many studiesand explorations on the design and construction of fusion proteins toimprove the activity of fusion proteins. such as changing the linkingorder of the fused proteins, changing different fusion sites, usingdifferent fusion partners, or using a peptide linker, etc.

Compared with other fusion strategies, the use of a peptide linker has avariety of advantages. First, the amino acids that make up the peptidelinker are diverse (20 common amino acids). The length of the peptidelinker is also an important tunable parameter, which can lead to richdiversity of peptide linkers (20^(n), n is the number of amino acidresidues of the peptide linker). The peptide linkers are easy forbioengineering modifications. Second, the peptide linker providescertain spatial spacing, such that the two fused proteins fold correctlywithout interfering with each other. Third, the peptide linker can alsoprovide more interaction possibilities for two fused proteins, promotingsynergic interactions.

There are currently two kinds of commonly used peptide linkers, helicalrigid peptide linkers (such as A(EAAAK)_(n)A (SEQ ID NO: 34)) andflexible peptide linkers comprising less hydrophobic and less chargedamino acids. The helical rigid peptide linkers can effectively separatedifferent functional regions of the fusion proteins. Examples offlexible peptide linkers include the (GGGGS)₃ sequence (SEQ ID NO: 35)that Huston designed. In addition, it has been reported that thecarboxyl terminal peptide (hereinafter referred to as CTP) of humanchorionic gonadotropin (HCG) beta chain can be used as a peptide linkeralone, and is mainly used to link different subunits of the sameprotein. For example, CTP is used as a peptide linker between the betaand alpha subunits of follicle stimulating hormone, as disclosed inChinese Patent Nos. CN103539860A, CN103539861A, CN103539868A andCN103539869A. In WO2005058953A2, CTP is used as a peptide linker in thefusion protein, linking the glycoprotein beta and alpha subunits.However, because CTP has the effect of prolonging the in vivo half-lifeof certain proteins, it has been primarily disclosed as a half-lifeprolonging moiety in the fusion proteins in many other patents. Thehalf-life prolonging moiety can optionally choose CTP, immunoglobulinFc, or other fusion partners with similar half-life prolonging function.

A number of literatures have reported the effects of the peptide linkersequences on the construction and expression of fusion proteins. Forexample, in the construction of the single chain antibody 1F7, it wasfound that using a conventional Genex212 peptide linker (GSTSGSGKSSEGKG(SEQ ID NO: 36)) to link the light and heavy chains did not achieve theoriginal catalytic activity of the protein and the fusion protein wasunstable. After a library of 18 amino acid residues with randomsequences was constructed and screened, catalytically active singlechain antibodies were obtained (Tang Y et al., 1996, J Biol Chem.,271:15682-15686). In studying the helical peptide, Arai et al. foundthat the fluorescence resonance energy transfer from EBFP to EGFPreduced as the length of the peptide linker increased, suggesting thatincreasing the linker length could effectively separate the twofunctional regions (Arai R. et al., 2001, Protein Eng., 14:529-532). Inaddition, in studying dengue virus NS2B protein, Luo et al. found thatinserting a glycine residue between the original sites 173 and 174, orreplacing proline 174 with glycine, resulted in a significant decreasein protein activity of both the N- and C-terminal proteins. Thisindicated that the length and rigidity of the native peptide linker werethe result of long-term evolution, and were of great significance to thefunction of the natural fusion proteins (Luo D. et al., 2010, J BiolChem., 285:18817-18827). The N-terminus of the NS2B protein was a serineprotease, the C-terminus was a RNA helicase, and the peptide linkerbetween them had 11 amino acid residues.

The present inventors found that the length and amino acid compositionof the peptide linker, the presence or absence of glycosylation sites,the compatibility between the peptide linker and the two activemolecules, and other factors, could affect the function and stability ofthe fusion proteins. The inventors believe that the peptide linkershould have the following characteristics: 1) can make the linkedproteins fold effectively into proper conformations, does not causemolecular dynamics change, and preferably comprises non-immunogenicnatural amino acids. 2) should have the ability to prevent proteaseattack. 3) should try to avoid mutual impacts of the two fused proteinson each other.

In the absence of clear guidelines for the design of peptide linkers,the present inventors have developed new peptide linkers for theconstruction of fusion proteins, based on the inventors' long-termresearch experience, particularly those in studying Fc fusion proteins.Amazingly, the peptide linkers have advantage for maintaining thebiological activity of proteins or polypeptides and have broadapplicability and portability.

DESCRIPTION OF THE INVENTION

The object of the present invention is to provide a novel peptide linkerfor constructing fusion proteins.

The first aspect of the present invention provides a peptide linker. Thepeptide linker comprises a flexible peptide and a rigid peptide. Theflexible peptide consists of one or more flexible units, and the rigidpeptide consists of one or more rigid units, wherein the flexible unitcomprises two or more amino acid residues selected from Gly, Ser, Alaand Thr, and the rigid unit comprises the carboxyl terminal peptide(CTP) of human chorionic gonadotropin β subunit.

Preferably, the peptide linker is glycosylated. More preferably, theglycosylation site is located on CTP. Much more preferably, theglycosylation process is accomplished by expression in mammalian cells,such as Chinese hamster ovary cells, or other expression hosts having asuitable glycosylation modification system.

Further, the rigid peptide in the peptide linker of the presentinvention is located at the N-terminus or C-terminus of the flexiblepeptide. Preferably, the rigid peptide is located at the C-terminus ofthe flexible peptide. Specifically, the structural formula of thepeptide linker of the present invention can be represented as F-R orR-F, wherein F and R represent the flexible peptide and the rigidpeptide, respectively. Further, the flexible peptide preferablycomprises 1, 2, 3, 4 or 5 flexible units, and the rigid peptidepreferably comprises 1, 2, 3, 4 or 5 rigid units.

More preferably, the flexible unit comprises two or more G and Sresidues. Much more preferably, the amino acid sequence of the flexibleunit has the general formula (GS)_(a)(GGS)_(b)(GGGS)_(c)(GGGGS)_(d) (SEQID NO: 37), wherein a, b, c and d represent the number of structuralunits composed of G and S residues, and are integers greater than orequal to 0, and a+b+c+d≥1.

Illustratively, each flexible unit is represented by F, and i=1, 2, 3,4, 5, . . . n. In some embodiments of the invention, the flexiblepeptide preferably comprises, but is not limited to the followingflexible units:

(i) F1: (SEQ ID NO: 21) GSGGGSGGGGSGGGGS; (ii) F2: (SEQ ID NO: 22)GSGGGGSGGGGSGGGGSGGGGSGGGGS; (iii) F3: (SEQ ID NO: 23)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; (iv) F4: (SEQ ID NO: 24)GSGGGGSGGGGSGGGGS; (v) F5: (SEQ ID NO: 25) GGGSGGGSGGGSGGGSGGGS;(vi) F6: (SEQ ID NO: 26) GGSGGSGGSGGS.

The rigid unit is selected from the full length or fragment sequence ofthe C-terminal amino acids 113 to 145 of human chorionic gonadotropin βsubunit. Specifically, the rigid unit comprises the amino acid sequenceof SEQ ID no: 1 or its truncated form.

Preferably, CTP contains at least 2 glycosylation sites. For example, ina preferred embodiment of the present invention, CTP contains 2glycosylation sites. Illustratively, CTP contains the N-terminal 10amino acids of SEQ ID no: 1, i.e. SSSS*KAPPPS* (residues 6-15 of SEQ IDNO: 1), or CTP contains the C-terminal 14 amino acids of SEQ ID no: 1,i.e. S*RLPGPS*DTPILPQ (residues 20-33 of SEQ ID NO: 1). For anotherexample, in another embodiment of the present invention, CTP contains 3glycosylation sites. Illustratively, CTP contains the N-terminal 16amino acids of SEQ ID no: 1, i.e. SSSS*KAPPPS*LPSPS*R (residues 6-21 ofSEQ ID NO: 1). For other examples, in other embodiments of the presentinvention, CTP contains 4 glycosylation sites. Illustratively, CTPcontains 28, 29, 30, 31, 32, or 33 amino acids, starting from position113, 114, 115, 116, 117, or 118 and ending at position 145 of the humanchorionic gonadotropin beta subunit. Specifically, CTP contains theN-terminal 28 amino acids of SEQ ID no: 1, i.e.SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (residues 6-33 of SEQ ID NO: 1). Inthis text, * represents a glycosylation site. Each possibilityrepresents a separate embodiment of the present invention.

In other embodiments, the rigid units provided by the present inventionhave at least 70% amino acid sequence identity to native CTP. In otherembodiments, the rigid units provided by the present invention have atleast 80% amino acid sequence identity to native CTP. In otherembodiments, the rigid units provided by the present invention have atleast 90% amino acid sequence identity to native CTP. In otherembodiments, the rigid units provided by the present invention have atleast 95% amino acid sequence identity to native CTP.

Illustratively, each rigid unit is represented by R_(i) and i=1, 2, 3,4, 5, . . . n. The rigid peptides described in some embodiments of thepresent invention may preferably comprise, but are not limited to, thefollowing CTP rigid units.

(i) R1: (SEQ ID NO: 27) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (ii) R2:(SEQ ID NO: 28) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (iii) R3:(SEQ ID NO: 29) SSSSKAPPPS; (iv) R4: (SEQ ID NO: 30) SRLPGPSDTPILPQ;(v) R5: (SEQ ID NO: 31) SSSSKAPPPSLPSPSR.

The rigid peptides of the present invention may further comprise two orthree of the above CTP rigid units. In one embodiment of the presentinvention, the rigid peptide comprises two R3 rigid units:SSSSKAPPPSSSSSKAPPPS (SEQ ID NO: 32) (represented as R3R3). In anotherembodiment, the rigid peptide comprises three R4 rigid units:SRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ (SEQ ID NO: 33) (representedas R4R4R4).

In some preferred embodiments of the invention, the peptide linker hasthe amino acid sequence as shown in SEQ ID no: 2

(represented as F2-R1) (GSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ).

In another preferred embodiment of the present invention, the peptidelinker has the amino acid sequence as shown in SEQ ID no: 3

(represented as F1-R2) (GSGGGSGGGGSGGGGSPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ).

In another preferred embodiment of the present invention, the peptidelinker has the amino acid sequence as shown in SEQ ID no: 4

(represented as F4-R1) (GSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILP Q).

In another preferred embodiment of the present invention, the peptidelinker has the amino acid sequence as shown in SEQ ID no: 5

(represented as F5-R5) (GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR).

In another preferred embodiment of the present invention, the peptidelinker has the amino acid sequence as shown in SEQ ID no: 6

(represented as F5-R5) (GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR).

In another preferred embodiment of the present invention, the peptidelinker has the amino acid sequence as shown in SEQ ID no: 7

(represented as F6-R4R4R4) (GGSGGSGGSGGSSRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ).

Yet another aspect of the invention provides a fusion protein containingthe peptide linker. The fusion protein comprises two biologically activemolecules and a peptide linker linking the two active molecules. Thestructural formula of the fusion protein is expressed as K1-L-K2 orK2-L-K1, wherein K1 is the first biologically active molecule, L is theabove-mentioned peptide linker, and K2 is the second biologically activemolecule. The components of the fusion protein are sequentially linkedfrom the N-terminus to C-terminus. Further, the active molecules may beselected from protein or protein domain, polypeptide, antibody orantibody fragment, preferably protein or protein domain, antibody or anantibody fragment.

Illustratively, the active molecule K1 of the fusion protein comprisesprotein or protein domain having biological function, polypeptide (e.g.,especially soluble or membrane signal molecule), cytokine, growthfactor, hormone, costimulatory molecule, enzyme, receptor, protein orpolypeptide having ligand function for the receptor. The active moleculeK2 is serum protein or protein domain that prolongs the circulationhalf-life, for example, human serum albumin (HSA), transferrin (TF),antibody/immunoglobulin Fc fragment, and so on.

Illustratively, the active molecule K1 of the fusion protein comprisestoxin, enzyme, cytokine, membrane protein, or immunomodulatory cytokine.The active molecule K2 comprises antibody or antibody fragment. K1 andK2 are linked via the peptide linker to form an antibody fusion protein.For example, K2 is an antibody Fv fragment (VL or VH), and for anotherexample, K2 is a single chain antibody (scFv).

Further, the biologically active molecule K1 comprises but is notlimited to adenosine A1 receptor, angiotensin converting enzyme ACE,activin family, ADAM family, ALK family, α-1-antitrypsin, programmedcell death associated protein family, nerve growth factor and receptorfamily, bone morphogenetic protein BMP and receptor family, complementfactor, calcitonin, cancer associated antigen, cathepsin family, CCLchemokine and receptor family, CD superfamily, CFTR, CXCL chemokine andreceptor family, EGF, epidermal growth factor EGF and receptor family,coagulation factor IIa, factor VII, factor VIII, factor IX, ferritin,fibroblast growth factor FGF and receptor family, follicle stimulatinghormone, FZD family, HGF, glucagon, cardiac myosin, growth hormone, Ig,IgA receptor, IgE, insulin-like growth factor IGF and binding proteinfamily, interleukin IL superfamily and its receptor superfamily,interferon INF family, iNOS, integrin family, kallikrein family,laminin, L-selectin, luteinizing hormone, MMP family, mucin family,cadherin superfamily, platelet-derived growth factor PDGF and receptorfamily, parathyroid hormone, serum albumin, T-cell-related receptorsuperfamily, TGF-α, transforming growth factor TGF-β superfamily,thyroid stimulating hormone, parathyroid stimulating hormone, tumornecrosis factor TNF superfamily and its receptor TNFRSF superfamily,urokinase, WNT signaling pathway family, thymosin α1, thymosin β4, VEGF,vascular endothelial growth factor VEGF and its receptor family.

In the preferred embodiments of the present invention, the activemolecule K1 is human coagulation factor VII (FVII), human coagulationfactor VIII (FVIII), GLP-1 analogue Exendin-4 (Ex4), human interleukin 7(IL-7), or human growth hormone (hGH). The peptide linker L of thefusion protein is as shown in SEQ ID no: 2, 3, 4, 5, 6 or 7. The activemolecule K2 is selected from the Fc fragments of human immunoglobulinsIgG, IgM, and IgA. More preferably, the Fc fragments are derived fromhuman IgG₁, IgG₂, IgG₃ or IgG₄. Furthermore, the Fc fragments may bewild-types or variants. The Fc variants contain at least one amino acidmodification in the wild-type human immunoglobulin Fc domain. Thevariants have reduced effector functions (ADCC or CDC effect) and/or anenhanced binding affinity for the neonatal receptor FcRn. Further, inthe preferred embodiments of the present invention, the Fc variants arepreferably selected from the following groups: (i) vFcγ1: human IgG₁hinge, CH2, and CH3 regions containing Leu234Val, Leu235Ala andPro331Ser mutations (the amino acid sequence is as shown in SEQ ID no:8); (ii) vFcγ2-1: human IgG₂ hinge, CH2, and CH3 regions containingPro331Ser mutation (the amino acid sequence is as shown in SEQ ID no:9); (iii) vFcγ2-2: human IgG₂ hinge, CH2, and CH3 regions containingThr250Gln and Met428Leu mutations (the amino acid sequence is as shownin SEQ ID no: 10); (iv) vFcγ2-3: human IgG₂ hinge, CH2, and CH3 regionscontaining Pro331Ser, Thr250Gln and Met428Leu mutations (the amino acidsequence is as shown in SEQ ID no: 11); (v) vFcγ4: human IgG₄ hinge,CH2, and CH3 regions containing Ser228Pro and Leu235Ala mutations (theamino acid sequence is as shown in SEQ ID no: 12).

More preferably, in one embodiment of the present invention, the FVII-Fcfusion protein comprises, sequentially from the N- to C-terminus, FVII(having the amino acid sequence as shown in SEQ ID no: 13), the peptidelinker (having the amino acid sequence as shown in SEQ ID no: 2), andhuman IgG Fc (having the amino acid sequence as shown in SEQ ID no: 11).

More preferably, in one embodiment of the present invention, theFVIII-Fc fusion protein comprises, sequentially from the N- toC-terminus, FVIII (having the amino acid sequence as shown in SEQ ID no:14), the peptide linker (having the amino acid sequence as shown in SEQID no: 2), and human IgG Fc (having the amino acid sequence as shown inSEQ ID no: 11).

More preferably, in one embodiment of the present invention, theExendin-4-Fc fusion protein comprises, sequentially from the N- toC-terminus, Exendin-4 (having the amino acid sequence as shown in SEQ IDno: 15). the peptide linker (having the amino acid sequence as shown inSEQ ID no: 2, 3, 5 or 7), and human IgG Fc (having the amino acidsequence as shown in SEQ ID no: 11).

More preferably, in one embodiment of the invention, the IL-7-Fc fusionprotein, sequentially from the N- to C-terminus, comprises IL-7 (havingthe amino acid sequence as shown in SEQ ID no: 16), the peptide linker(having the amino acid sequence as shown in SEQ ID no: 2), and human IgGFc (having the amino acid sequence as shown in SEQ ID no: 11).

More preferably, in one embodiment of the present invention, the hGH-Fcfusion protein comprises, sequentially from the N- to C-terminus, hGH(having the amino acid sequence as shown in SEQ ID no: 17), the peptidelinker (having the amino acid sequence as shown in SEQ ID no: 2), andhuman IgG Fc (having the amino acid sequence as shown in SEQ ID no: 11).

In other preferred embodiments of the invention, the active molecule K1of the fusion protein is the antibody heavy chain variable region (VH),and K2 is the antibody light chain variable region (VL). K1 and K2 arelinked by the peptide linker to form a single chain antibody (scFv).

In some preferred embodiments of the invention, the active molecule K1of the fusion protein comprises a first antibody or antibody fragment,and the active molecule K2 comprises a second antibody or antibodyfragment. K1 and K2 are linked by the peptide linker to form abispecific antibody.

Preferably, in one embodiment of the present invention, K1 is afull-length double-chain anti-CD20 antibody, K2 is a single chainanti-CD3 antibody, and K1 and K2 are linked by the peptide linker toform a bispecific antibody. More preferably, the heavy chain of theanti-CD20 double-chain antibody contained in the bispecific antibody hasan amino acid sequence as shown in SEQ ID no: 18 and the correspondinglight chain has an amino acid sequence as shown in SEQ ID no: 19. Theanti-CD3 single chain antibody contained in the bispecific antibody hasan amino acid sequence as shown in SEQ ID no: 20. The peptide linker hasan amino acid sequence as shown in SEQ ID no: 4 or 6. The heavy chain ofthe anti-CD20 double-chain antibody is linked to the anti-CD3 singlechain antibody by the peptide linker.

Yet another aspect of the present invention also provides a method forpreparing a fusion protein having the structural formula expressed as:K1-L-K2 or K2-L-K1, wherein K1 is the first biologically activemolecule, L is the peptide linker, and K2 is a second biologicallyactive molecule. The components that make up the fusion protein aresequentially linked from the N- to the C-terminus. The active moleculesmay be selected from the groups including protein or protein domain,polypeptide, antibody or antibody fragment, preferably protein orprotein domain, antibody or antibody fragment. The preparation methodincludes the step of allowing K1 and K2 to be linked by L. In thepreferred embodiments of the present invention, the method includes thefollowing steps:

(a) Ligate the DNA sequences encoding the first active molecule K1 andthe second active molecule K2 through the DNA sequence of the peptidelinker L to form a fusion gene.

(b) Introduce the fusion gene obtained in step (a) into a eukaryotic orprokaryotic expression host.

(c) Culture the high yield expression host screened and obtained in step(b) to express the fusion protein.

(d) Harvest the fermentation broth of step (c) and isolate and purifythe fusion protein.

Illustratively, the peptide linkers are used to link the activeproteins/polypeptides to serum proteins with long circulationhalf-lives, such as antibody/immunoglobulin Fc fragments, human serumalbumin (HSA), transferrin (TF), etc. In one preferred embodiment of thepresent invention, the preparation method of the FVII-Fc fusion proteinincludes the step of linking the active molecule FVII and human IgG Fcby the peptide linker (SEQ ID no: 2). In one preferred embodiment of thepresent invention, the preparation method of the FVIII-Fc fusion proteinincludes the step of linking the active molecule FVIII and human IgG Fcby the peptide linker (SEQ ID no: 2). In another preferred embodiment ofthe present invention, the preparation method of the Exendin 4-Fc fusionprotein includes the step of linking the active molecule Exendin-4 andhuman IgG Fc by the peptide linker (SEQ ID no: 2, 3, 5 or 7). In anotherpreferred embodiment of the present invention, the preparation method ofthe IL-7-Fc fusion protein includes the step of linking the activemolecule IL-7 and human IgG Fc by the peptide linker (SEQ ID no: 2). Inanother preferred embodiment of the present invention, the preparationmethod of the hGH-Fc fusion protein includes the step of linking theactive molecule hGH and human IgG Fc by the peptide linker (SEQ ID no:2).

Illustratively, the peptide linkers are used in the construction ofbispecific antibodies. In the preferred embodiment of the presentinvention, the preparation method of the bispecific antibody ofanti-CD3XCD20 includes the step of linking the double-chain anti-CD20antibody and the single chain anti-CD3 antibody by the peptide linker(SEQ ID NO: 4 or 6).

In this present invention, technical novelities can be summarized asfollows:

1. In the present invention, part of the peptide linker, CTP, containsmultiple 0-glycosyl side chains. It is capable of forming a relativelystable and rigid three-dimensional structure and thus more effectivelyseparate the two partners of the fusion protein and eliminate the sterichindrance between them. In the construction of a series of fusionproteins composed of the active proteins and the Fc fragment, forexample, the FVII-Fc fusion protein, the introduction of the rigid CTPunit into the peptide linker ensures that the N-terminally fused activeprotein does not affect the binding site of the Fc variant to FcRn, thushaving no effect on the circulation half-life of the fusion protein. Inaddition, the protein A binding site of Fc is important for thepurification step, and the introduction of the rigid CTP unit ensuresthat the N-terminally fused active protein will not “block” the proteinA binding site. On the other hand, the introduction of the rigid CTPunit makes the 25 kD size of the Fc fragment not interfere with thefolding of the N-terminally fused active protein, which prevents thedecline or loss of the biological activity/function of the activeprotein. Many embodiments of the present invention indicate that theintroduction of the rigid CTP unit makes the biological activity of thefusion proteins significantly improved. This can be explained asfollows. The rigid CTP polypeptide possesses multiple glycosyl sidechains. Compared with irregular coil of a flexible peptide linker suchas (GGGGS)_(n) (SEQ ID NO: 38), CTP can form a stable three-dimensionalconformation, which effectively increases the distance between the twofusion partners of the fusion protein. The spatial separationfacilitates independent folding of the active protein and the Fc segmentto form correct three-dimensional conformations, such that the activeprotein and the Fc segment would have no mutual impact on the biologicalactivity of each other. This reduces the possibility of the decline orloss of the activity of the active protein due to misfolding andconformational alteration. Thus the biological activity of the fusionprotein is increased.

2. The peptide linkers of the present invention have a wide range ofapplicability and portability. With a combination of rigid and flexibleunits, the peptide linkers are conferred with a conformation betweencompletely rigid and fully flexible, and the specific rigidity orflexibility of the polypeptide varies depending on the ratio andarrangement order of the two sequences. As sequences are designed withdifferent combinations of ratio and arrangement order of rigid andflexible units, the rigidity of the peptide linker can be finelyregulated to meet different requirements in the construction of thefusion proteins.

3. CTP contains glycosylation sites. Highly sialylated and negativelycharged CTP can resist the kidney to its clearance and further prolongsthe half-life of the fusion protein. CTP can improve pharmacokineticparameters, such as reducing the clearance rate, reducing the apparentdistribution of volume, increasing AUC_((0-t)), such that thebioavailability of the fusion protein increases. It is expected that theclinical dose will be reduced.

4. The glycosyl side chains of CTP has protective effect, which canreduce the sensitivity of the peptide linker to proteases. The fusionprotein is not easy to be degraded in the linking region.

Term Definitions

“Antibody fragment”: refers to an antigen-binding fragment of anantibody or antibody analogue, which typically comprises at least aportion of the antigen-binding region or variable region of the parentalantibody, for example, one or more CDRs.

The “Fc” region: includes two heavy chain fragments, each of whichcomprises the CH2 and CH3 domains of an antibody. The two heavy chainfragments are held together by two or more interchain disulfide bondsand the inter-CH3 domain hydrophobic interactions.

The “Fv” region comprises the variable regions from both the heavy andlight chains, but lacks constant regions.

“Single chain Fv antibody” (or scFv antibody): refers to an antibodycomprising the VH and VL domains of an antibody, wherein these domainsare present in a single polypeptide chain. In general, the Fvpolypeptide additionally contains a polypeptide linker between the VHand VL domains that allows scFv to form the desired structure forantigen binding.

“Bispecific antibody”: refers to an antibody that comprises two Fvdomains or scFv units such that the resulting antibody recognizes twodifferent antigenic determinants

“Antibody fusion protein”: refers to a product obtained by fusing anantibody fragment with another bioactive protein using geneticengineering techniques. Owing to many different fused proteins, theantibody fusion proteins have a variety of biological functions.

For example, an Fv-containing antibody fusion protein. As the Fab or Fvfragment is linked with certain toxins, enzymes, or cytokines, thebiologically active molecules can be targeted to specific sites of thetargeted cell, forming the so-called “biological missile.

For example, chimeric receptors. As the scFv antibody is fused withcertain cell membrane protein molecules, the fusion proteins, known aschimeric receptors, can be expressed on the cell surface, giving thecells the capability to bind to a specific antigen.

For example, an Fc-containing antibody fusion protein. The antibody IgGFc region is fused with the biologically active molecule to form an Fcfusion protein. The Fc fusion protein not only exerts the biologicalfunction of the active molecule but also inherits similar properties ofthe antibody, including prolonged plasma half-life and a series ofeffector functions specific to the Fc region. For example, on the onehand, the Fc region plays an important role in the eradication ofpathogens. The Fc-mediated effector functions of IgG are carried out bytwo mechanisms: (1) After the Fc regions of IgG molecules bind to thecell surface Fc receptors (FcγRs), pathogens are broken down byphagocytosis or lysis or by killer cells through an antibody-dependentcell-mediated cytotoxicity (ADCC) pathway. (2) After the Fc regions ofIgG molecules bind to C1q molecules of the first complement C1complexes, the complement-dependent cytotoxicity (CDC) pathway istriggered, such that pathogens are lysed. On the other hand, theantibody Fc region bind to the FcRn receptor to prevent the antibodyfrom entering into lysosome to be degraded. The fusion proteinscontaining the Fc region are endocytosed and protected by FcRn. Thesefusion proteins are not to be degraded, but again enter into thecirculatory system, thereby increasing the in vivo half-lives of thesefusion proteins. Moreover, FcRn shows activity in adult epithelialtissues, and it is expressed in epithelial cells of the intestine,pulmonary trachea, nasal cavity, vagina, colon, and rectum. The fusionproteins containing the Fc region can effectively shuttle the epithelialbarrier through FcRn-mediated cell transduction.

“hCG-β carboxy terminal peptide (CTP)”: is a short peptide derived fromthe carboxyl terminus of human chorionic gonadotropin (hCG) betasubunit. Four kinds of reproduction-related polypeptide hormones,follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroidstimulating hormone (TSH), and human chorionic gonadotropin (hCG)comprise the same alpha subunit and respective specific beta subunits.Compared with the other three hormones, hCG has a significantlyprolonged half-life, which is mainly due to the specific carboxylterminal peptide (CTP) on its β-subunit. CTP has 37 amino acid residues,which possesses four O-glycosylation sites terminating with a sialicacid residue. Highly sialylated, negatively charged CTP can resist tothe clearance by the kidney, thereby prolonging the in vivo half-life ofa protein (Fares F A et al., 1992, Proc Natl Acad Sci USA, 89:4304-4308).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Comparison of the bleeding times of HA mice administered withFP-A1 and NovoSeven® for 1 h and 2 h. Compared with the HA-N-1h group,*P<0.05, ***P<0.01; compared with the C57-NS group, ^(#)P<0.05,^(###)P<0.01.

FIG. 2. The active half-lives of FP-A1 and NovoSeven® in ratsadministered with warfarin.

FIG. 3. The curves of the RBG value changes in 0-216 h in db/db diabeticmice given single injection of FP-C1 and FP-C5.

FIG. 4. The effect of different doses of FP-C1 on HbA1c in db/dbdiabetes mice. Comparison of the FP-C1 group with the model group,*P<0.05, **P<0.01.

FIG. 5. RBG value changes of STZ-induced diabetes mice with singleinjection of FP-C1 during 0-240 h. Comparison of the FP-C1 group withthe modle group, *P<0.05, **P<0.01; Comparison of the Duraglutide groupwith the module group, 2P<0.05, 2P<0.01.

FIG. 6. The effect of FP-C1 on the body weight of mice fed with high fatdiet.

FIG. 7. The effect of FP-C1 on glucose tolerance in mice fed with highfat diet (mean±SD, n=8). Compared with the normal group, #P<0.05,##P<0.01; compared with the high fat group, *P<0.05, **P<0.01.

FIG. 8. The effect of FP-C1 on the serum insulin concentration of micefed with high fat diet (means±SD, n=8). Compared with the normal group,#P<0.05, ##P<0.01; compared with the high fat group, *P<0.05, **P<0.01.

FIG. 9. The effect of FP-C1 on the insulin resistance index of mice fedwith high fat diet (mean±SD, n=8). Compared with the normal group,#P<0.05, ##P<0.01; compared with the high fat group, *P<0.05, **P<0.01.

FIG. 10. The effect of FP-C1 on the cross-sectional area of fat cells inmice fed with high fat diet. A: normal group. B: high fat diet group. C:FP-C1 group.

FIG. 11. The ability of IL7-Fc fusion proteins FP-D1 and FP-D2 tostimulate mouse mononuclear cells to proliferate.

FIG. 12. The ability of hGH-Fc fusion proteins FP-E1 and FP-E2 tostimulate Nb2 cells to proliferate.

FIG. 13. The curves of blood drug concentration vs. time of hGH-Fcfusion proteins FP-E1 and FP-E2.

FIG. 14. The growth curves of all groups of rats administered with thehGH-Fc fusion protein FP-E1.

FIG. 15. The anti-CD3×CD20 bispecific antibody FP-F1 activated humanPBMC cells to secret IFN-γ in a concentration-dependent manner.

FIG. 16. The anti-CD3×CD20 bispecific antibody FP-F1 activated humanPBMC cells in a concentration-dependent manner.

FIG. 17. The efficacy of anti-CD3×CD20 bispecific antibody FP-F1 inkilling subcutaneous transplantation tumor.

EXAMPLES Example 1. Peptide Linkers Used to Construct Fusion Proteins

The present inventors constructed a series of fusion proteins K1-L-K2containing peptide linkers. The composition of each fusion protein wasshown in Table 1. The DNA sequences encoding the first active moleculeK1 and the second active molecule K2 were ligated by the DNA sequence ofthe peptide linker L to constitute a fusion gene. Preferably, the codonsof the DNA sequences were optimized for expression in CHO cells.Preferably, the sequences were generated by chemical synthesis. Tworestriction sites, SpeI and EcoRI, were added at the 5′ and 3′ ends ofthe synthesized fragment, respectively, to facilitate insertion of thefusion gene obtained above into the specific site of an expressionvector. After verified by sequencing, the fusion gene was digested withthe corresponding restriction endonucleases and inserted into thecorresponding cleavage sites of the expression plasmid PXY1A1, obtainedby modifying PCDNA3.1 as a template. The expression plasmid containingthe gene of the fusion protein was thus obtained. The PXY1A1 plasmidcontained, but was not limited to, the following important expressionelements: 1) human cytomegalovirus immediate early promoter and highlyexogenous expression enhancer needed by mammalian cells; 2) doublescreening markers with kanamycin resistance in bacteria and G418resistance in mammalian cells; 3) murine dihydrofolate reductase (DHFR)gene expression cassette. When the host cell type was DHFR genedeficient, methotrexate (MTX) could co-amplify the fusion gene and theDHFR gene (U.S. Pat. No. 4,399,216). The expression plasmid of thefusion protein was transfected into a mammalian host cell line. Thepreferred host cell line was DHFR enzyme-deficient CHO cells in order toachieve stable and high levels of expression (U.S. Pat. No. 4,818,679).After two days of transfection, the medium was replaced with a screeningmedium containing 0.6 mg/mL G418. Cells were seeded in the 96-well plateat a certain concentration (5000-10000 viable cells/well) for 10-14 daysuntil large discrete cell clones appeared. The transfectants resistantto the selecting antibiotic were screened by the ELISA assay. Subcloneswith high level expression of the fusion protein were isolated bylimiting dilution and use of the 96-well culture plate. For a fusionprotein already validated as a good one by multiple means, it wasappropriate to amplify the DHFR gene by MTX drug inhibition to achievehigher level of expression. In the growth medium containing increasingconcentration of MTX, the transfected fusion protein gene wasco-amplified with the DHFR gene. The highly expressed monoclonal cellstrains obtained were cultured under fed-batch conditions in shakeflasks or a 5-liter fermentor. The fusion protein was purified byprotein A affinity chromatography and other ion exchange chromatography.

The inventors constructed a series of fusion proteins containing thepresent invention's peptide linkers with flexible and rigid units, andalso constructed a variety of fusion proteins containing the peptidelinkers with only flexible units of different lengths for comparison.For example, FVII-Fc fusion protein (FP-A1 containing CTP; FP-A2 andFP-A3 without CTP), FVIII-Fc fusion protein (FP-B1 containing CTP; FP-B2and FP-B3 without CTP); Fc-fusion protein of Exendin-4 and its analogue(FP-C1, FP-C2, FP-C3, FP-C4 containing CTP; FP-05 without CTP), IL7-Fcfusion protein (FP-D1 containing CTP; FP-D2 without CTP), hGH-Fc fusionprotein (FP-E1 containing CTP; FP-E2 without CTP). In addition, theinventors also developed anti-CD20×CD3 bispecific antibodies (FP-F1 andFP-F2 containing CTP). The composition of each fusion protein was shownin Table 1, and the amino acid sequence of each fusion protein was shownin the sequence listings section.

TABLE 1 The compositions of various fusion proteins from N- toC-terminus Code for fusion protein K1 L K2 FP-A1 FVII F2-R1 Fc (vFcγ₂₋₃)FP-A2 FVII F1 Fc (vFcγ₂₋₃) FP-A3 FVII F3 Fc (vFcγ₂₋₃) FP-B1 FVIII F2-R1Fc (vFcγ₂₋₃) FP-B2 FVIII F1 Fc (vFcγ₂₋₃) FP-B3 FVIII F3 Fc (vFcγ₂₋₃)FP-C1 Exendin-4 F2-R1 Fc (vFcγ₂₋₃) FP-C2 Exendin-4 F1-R2 Fc (vFcγ₂₋₃)FP-C3 Exendin-4 F5-R5 Fc (vFcγ₂₋₃) FP-C4 Exendin-4 F6-R4R4R4 Fc(vFcγ₂₋₃) FP-C5 Exendin-4 F1 Fc (vFcγ₂₋₃) FP-D1 IL-7 F2-R1 Fc (vFcγ₂₋₃)FP-D2 IL-7 F1 Fc (vFcγ₂₋₃) FP-E1 hGH F2-R1 Fc (vFcγ₂₋₃) FP-E2 hGH F1 Fc(vFcγ₂₋₃) FP-F1 Anti-CD20 mAb F4-R1 Anti-CD3 ScFv FP-F2 Anti-CD20 mAbF4-R3R3 Anti-CD3 ScFv

Example 2. Preparation of Coagulation Factor FVII-Fc Fusion Proteins andDetermination of Biological Activity and In Vivo Active Half-life

2.1 Preparation and Identification of Coagulation Factor FVII-Fc FusionProteins

The stably expressing CHO cell strains of FP-A1, FP-A2 and FP-A3obtained in Example 1 were cultured in shake flasks under fed-batchconditions for 10-14 days. The fusion proteins were purified by foursteps of column chromatography: protein A affinity chromatography,multidimensional chromatography, anion exchange chromatography, andmolecular sieve chromatography. The fusion proteins were thenself-activated with incubation in a solution. The SDS-PAGEelectrophoresis showed the following results. Under reducing conditions,the single-chained molecule of non-activated FP-A2 showed two obviousbands in the vicinity of 70-85 kDa and 40 kDa, indicating that proteindegradation occurred and the fraction of the degraded fragments wasabout 20-30%. Under non-reducing conditions, the non-activated FP-A2migrated to about 130 kDa together with another band of >200 kDa,indicating that some of the fusion proteins aggregated. Under reducingconditions, the single-chained molecule of non-activated FP-A3 was closeto 100 kDa and there were no contaminant bands. Under non-reducingconditions, most of the non-activated FP-A3 proteins migrated to >200kDa, indicating that FP-A3 was in the form of aggregates. Under reducingconditions, the single-chained molecule of non-activated FP-A1 was of100-110 kDa and there were no obvious contaminant bands. Undernon-reducing conditions, the non-activated FP-A1 migrated to 150 kDa.Under reducing conditions, the activated FP-A1 showed two clear bands,74.3 kDa HC-L-CTP-Fc and about 24.0 kDa LC, respectively, and no othercontaminant bands. Under non-reducing conditions, the activated FP-A1migrated to 150 kDa, indicating that the fusion protein FP-A1 did notdegrade significantly, did not form aggregates remarkably, and hadhigher thermodynamic stability and stronger ability of anti-proteolytichydrolysis. This example demonstrated that the peptide linkerscontaining the rigid CTP unit increased the stability of the fusionproteins, which were less susceptible to degradation, and reduced theformation of aggregates.

2.2 Direct Determination of the Biological Activity of Fusion Proteinsby the Clotting Assay

The determination of FVIIa biological activity by the clotting assay wasachieved by correcting FVIIa-deficient plasma to prolong the clottingtime. The kit (STAGO, Cat. No. 00743) was used for the assay. The assaywas first to mix diluted human freeze-dried normal plasma of known FVIIactivity (Unicalibrator, Cat. No. 00625) with FVII-deficient plasma,measure the prothrombin time (PT), and establish a standard curve. Theplasma for the FVII activity to be measured was diluted and then mixedwith FVII-deficient plasma for PT measurement. The FVIIa activity of thetest sample could be determined from the logarithmic equation betweenthe percentage of activity C (%) and PT time t (s) fitted by thestandard curve. The FVIIa activity was expressed as the percentage (%)of that of normal plasma. The corresponding relation between thepercentage of activity of the standards (%) provided by the kit and theinternational unit (IU) of enzyme activity was 100%=1 IU, according towhich the specific activity of FVII in the test sample could becalculated in units of IU/mg. The results showed that under optimumexperimental conditions, the highest activities of FP-A 1, FP-A2 andFP-A3 were about 20000 IU/mg, 4000 IU/mg and 7000 IU/mg, respectively.The experimental results showed that the type and length of the peptidelinkers had a great effect on the activity and stability of FVII-Fc. Thein vitro bioactivity of the fusion protein FP-A1 containing the peptidelinker with the rigid unit was much higher than those of the fusionproteins FP-A2 and FP-A3 without rigid units. The results also showedthat if only the length of the flexible peptide was extended, theactivity of the fusion protein could not be improved effectively. Thesteric hindrance between the fused partners comprised the formation ofthe correct conformation of the fusion proteins, such that the stabilityof the fusion proteins decreased and the proteins were easy to formaggregates. This example demonstrated that the peptide linker containinga rigid CTP unit reduced the steric hindrance of the Fc domain andincreased the activity and stability of the fusion protein.

2.3 Determination of In Vivo Bleeding Inhibition of FVII-Fc FusionProteins

The hemostatic effect of FVII-Fc fusion proteins was assessed by usinghemophilia mice, which were the tail vein transection (TVT) bleedingmodel of homozygous hemophilia A mice with knockout of the FVIII factorgene (HA, Shanghai Research Center of the Southern Model Organisms).Male HA mice, 16-20 weeks old, were adaptively fed for one week andrandomly divided into 3 groups, 6 mice per group. Two groups were given300,000 IU/kg of FP-A1 and the other group was given 300,000 IU/kg ofNovoSeven® (Novo Nordisk). Meantime, wild-type male C57BL/6J mice, 16-20weeks old (Shanghai Research Center of the Southern Model Organisms),were used as the normal control group (n=6), and given an equal volumeof physiological saline through tail vein injection. The two groups ofHA mice given FP-A1 were subjected to tail-cutting tests at 1 h and 2 hafter administration, respectively, and the HA mice given NovoSeven®were subjected to tail-cutting tests at 1 h after administration. TheC57BL/6J normal control group (C57-NS group) was subjected totail-cutting tests at 2 h after administration. All data were expressedas mean±standard error (±SEM). The t-test analysis was used to comparebetween the experimental groups. The analysis software was GraphpadPrism 5.0. P<0.05 was considered statistically significant.

As shown in FIG. 1, after administration of NovoSeven® for 1 h, thebleeding time of the mice was 30 min, indicating that it had nohemostatic effect (HA-N-1h group). In contrast, after administration ofFP-A1 for 1 h (HA-F-1h group) and 2 h (HA-F-2h group), FP-A1 was stilleffective in hemostasis, and the bleeding times were significantlyshorter than that of the NovoSeven® group (P<0.05). This indicated thatFP-A1 had a significantly longer active half-life compared withNovoSeven®.

2.4 Determination of In Vivo Active Half-Life of FVII-Fc Fusion Protein

In this study, we investigated the active half-life of FP-A1 in thewarfarin-induced coagulation disorder rat model. According to the methodreported in the literature (Joe Salas et al., 2015, Thrombosis Research,135:970-976 or Gerhard Dickneite et al., 2007, Thrombosis Research,119:643-651), SD rats (8-12 weeks age and 220 g body weight, BeijingVital River Laboratory Animal Technology Co., Ltd.) were randomlydivided into two groups, eight rats per group. After intragastricadministration of warfarin (Orion Corporation, Finland, lot no. 1569755)at a dose of 2.5 mg/kg for 24 h, the rats were given intravenousadministration of 10,000 IU/kg of FP-A1 or NovoSeven® (Novo Nordisk),respectively. Blood was collected after administration for the FP-A1group at 0.05 h, 0.5 h, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, respectively, andfor the NovoSeven® group at 0.05 h, 0.5 h, 1 h, 2 h, 3 h, 5 h,respectively. With sodium citrate at a final concentration of 0.013 M asan anticoagulant, blood samples were centrifuged at 3000 rpm for 10 minto obtain the supernatant. The activity was determined by the method insection 2.2 and the active half-life was calculated.

As shown in FIG. 2, the active half-life of FP-A1 was calculated to be3.03±0.35 h, and that of NovoSeven® was 1.01±0.16 h. Compared withNovoSeven® with equal activity, FP-A1 prolonged the active half-life inrats by about 3-fold. The plasma coagulation activity was about 40% at 3h after single injection of FP-A1, while the coagulation activity ofNovoSeven® decreased to 3% after 3 h. At 12 h after administration, theplasma coagulation activity of FP-A1 remained 7% or more.

The results in sections 2.3 and 2.4 showed that the fusion protein ofthe present invention, containing the peptide linker with the flexibleand rigid CTP units, had a significantly prolonged active half-life,indicating that the peptide linker eliminated the blocking effect of theactive protein FVII on the binding site of Fc for its receptor FcRn. Theresults also demonstrated that owning to the introduction of the peptidelinker of the present invention, FVII formed the correctthree-dimensional conformation, maintained high biological activity, andwas not affected by the steric hindrance of the C-terminally fused Fc.

Example 3. Production of Coagulation Factor FVIII-Fc Fusion Proteins andDetermination of Biological Activity

3.1 Production and Identification of Coagulation Factor FVIII-Fc FusionProteins

The stably expressing CHO cell strains for FP-B1, FP-B2 and FP-B3obtained in Example 1 were cultured for 7 to 12 days under fed-batch orsemi-continuous culture conditions. The supernatant was harvestedimmediately for purification by protein A and/or VIII-select (GE)affinity chromatography. Under optimum culture conditions, the FP-B2supernatant was passed through protein A and VIII-select (GE) two-stepaffinity chromatography columns, and the elute still contained multiplecomponents. Under reducing conditions, the SDS-PAGE electrophoresisshowed a main band of 180 kDa and multiple fragments of 40-100 kDa.Under non-reducing conditions, most of the purified proteins migratedto >300 kDa. This indicated that FP-B2 products were mostly in the formof aggregates, unstable and easy to be degradated. Under the sameculture conditions, the supernatant of FP-B1 was purified by protein Aand VIII-select (GE) two-step affinity chromatography. Under reducingconditions, three clear bands appeared on the gel, which were singlechain FVIII-Fc (190 kDa), light chain-Fc (105 kDa) and heavy chain (90kDa), and there were no contaminant bands. Under non-reducingconditions, purified proteins FP-B1 and FP-B3 migrated to >200 kDa,while most of FP-B3 proteins stayed in the stacking gel, indicating thatFP-B3 was also present in the form of aggregates.

It was reported that the lipid binding region of FVIII (2303-2332) wascritical to its function, and that very small conformational changes inthe region led to protein aggregation and loss of activity (Gilbert etal., 1993, Biochemistry, 32:9577-9585). The results of the presentinvention indicated that the peptide linker containing the rigid CTPunit could eliminate the steric hindrance of the C-terminal Fc on theFVIII lipid-binding region, such that the FVIII spatial conformation wasalmost unaffected. Thus, the protein aggregation was reduced, theprotein stability was increased, and the bioactivity of the FVIII-Fcfusion protein was greatly improved.

3.2 Direct Determination of the Biological Activity of the FVIII-FcFusion Proteins by the Clotting Assay

The determination of the FVIII biological activity by the clotting assaywas achieved by correcting FVIII-deficient plasma to prolong theclotting time. A factor VIII (FVIII) assay kit (the clotting method),STA®-Deficient FVIII (STAGO, Cat. No. 00725), was used. The method wasfirst to determine the activated partial thromboplastin time (APTT) ofnormal human freeze-dried plasma (Unicalibrator, Cat No. 00625) of knowncoagulation factor VIII activity. The test instrument was a STAGO START®hemostasis analyzer. A standard curve was established. Then the FVIII-Fcfusion protein was mixed with the factor VIII-deficient plasma, and APTTvalues were determined. The logarithmic equation between the percentageof activity C (%) and APTT time t (s) fitted by the standard curve couldbe used to determine the activity of the test sample FVIII-Fc. Theactivity was expressed as the percentage (%) of that of normal plasma.The corresponding relation between the percentage of activity of thestandards provided by the assay kit and the international unit (IU) ofenzyme activity was 100%=1 IU, according to which the specific activityof FVIII in the test sample could be calculated, expressed as IU/mg. Theresults showed that under optimum experimental conditions, the highestactivities of FP-B1, FP-B2 and FP-B3 were 10000 IU/mg, 150 IU/mg and1300 IU/mg, respectively. Considering that most of the FP-B2 proteinmolecules were in the form of inactive aggregates or degraded fragments,the actual specific activities of FP-B2 and FP-B3 that were in activeforms were not necessarily very different. This indicated that extendingthe length of the flexible peptide linker had a limited effect onimproving the activity of the fusion protein FVIII-Fc. FP-B1 and FP-B2showed a significant difference between the specific activities, whichindicated that the rigid CTP unit in the peptide linker could reduce thesteric hindrance of the Fc domain and improved the activity of theFVIII-Fc fusion protein.

Example 4. Production of Exendin-4-Fc Fusion Proteins and Determinationof Biological Activity and In Vivo Active Half-Life

4.1 In Vitro Biological Activity

The stably expressing CHO cell strains of FP-C1, FP-C2, FP-C3, FP-C4 andFP-05 obtained in Example 1 were cultured in shake flasks underfed-batch conditions for 12-14 days. The fusion proteins were purifiedby Protein A affinity chromatography, and used for activity analysis.The purity of the fusion proteins was above 95%, and the molecular sizeswere as expected. For the in vitro activity assay, referred to theliterature (Zlokarnik G et al, 1998, Science, 279:84-88). The method wasbriefly described as follows. First, the human GLP-1R expression plasmidand the expression plasmid PGL-4.29 (Luc2P/CRE/Hygro) (Promega) carryingthe CRE-Luc reporter gene were co-transfected into CHO-K1 cells. Thenstable cell strains co-expressing both the plasmids were obtained afterscreening by antibiotic pressure. For the in vitro activity assay, 16000cells per well in 200 μL medium were inoculated into the 96-well cellculture plate. The cells were cultured in DMEM medium containing 10% FBSfor 16-24 h, until they grew to cover more than 90% of the bottom of thewells. The fusion proteins FP-C1, FP-C2, FP-C3, FP-C4 and FP-05 werediluted with DMEM medium containing 10% FBS, and 10 μL was added to eachwell of the 96-well culture plate. The concentration gradients were setto 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, and 2.5 nM.Meantime an equal concentration gradient of duraglutide (Eli Lilly, Cat.No. 9301897) was set as positive control. After the cells were incubatedat 37° C., 5% CO₂ for 5-6 h, the supernatant was aspirated. The cellswere washed slowly by adding 300 μL of PBS/well, then PBS was aspirated.40 μL of lysis buffer was added and shaken for 15 min, and then 40 μL ofluciferase substrate (Genomeditech (Shanghai) Co., Cat. No. GM-040501B,luciferase reporter gene detection kit) was added to each well. After 2min reaction time, the fluorescence was measured at a wavelength of 560nm using a multi-function microplate reader (SpectraMax M5 system,Molecular Device). A dose response curve was plotted based on thefluorescence values. The EC₅₀ value was calculated. The results wereshown in Table 2. The EC₅₀ values of FP-C1, FP-C2, FP-C3, FP-C4 andFP-05 were 0.03086 nM, 0.03156 nM, 0.03684 nM, 0.04012 nM and 0.03586nM, respectively, and that of duraglutide was 0.02987 nM. The in vitrobiological activities of CTP-containing FP-C1, FP-C2, FP-C3, FP-C4 andCTP-free FP-05 were comparable. As the inventors understood, for asimple small molecule polypeptide such as Exendin-4, the sterichindrance of Fc to Exendin-4 was small, the effect of CTP thateliminated the steric hindrance and increased the activity of the fusionprotein was not evident.

TABLE 2 Comparison of in vitro activity EC₅₀ values of fusion proteinsFusion protein FP-C1 FP-C2 FP-C3 FP-C4 FP-C5 Duraglutide EC₅₀ (nM)0.03086 0.03156 0.03684 0.04012 0.03586 0.02987

4.2 Blood Glucose Concentration Changes in Db/Db Diabetic Mice GivenSingle Injection of FP-C1 or FP-C5

Female diabetic db/db mice (Shanghai SLAC Laboratory Animals Co., Ltd.),8 weeks old, weighing 42±2 g, were randomly divided into 3 groups, with6 mice per each group. The drug-testing groups were injectedsubcutaneously with FP-C1 or FP-05 at a dose of 3 mg/kg. The positivegroup was injected with 3 mg/kg of duraglutide (Eli Lilly, Cat. No.9301897). The control group was injected with an equal volume of PBSbuffer (10 mL/kg). Blood samples were collected from the tail vein at 0h (before administration), 1 h, 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, 96 h,120 h, 144 h, 168 h, 192 h, 216 h (after administration). The bloodglucose meter was used to measure random blood glucose (RBG)concentrations for recording the data. The blood glucose data wereexpressed as mean±standard deviation (mean±SD) and analyzed using theSPSS 18.0 statistical software package.

At the same dose, FP-C1, FP-05 and the positive control drug duraglutideall had significant hypoglycemic effects. FIG. 3 showed the curves of9-day mouse RBG values after administration. The hypoglycemic effect ofduraglutide could only be maintained until day 4 (P>0.05). At 120 hafter administration, the blood glucose level had no statisticaldifference compared with that of the control group. In contrast, FP-C1could lower the blood glucose level of the mice for 168 h afteradministration, that was, the blood glucose level of the mice on the 7thday after administration was still statistically different from that ofthe control group (P<0.05). FP-05 could maintain the hypoglycemic effectfor 144 h after administration, that was, on the 6th day afteradministration the blood glucose level of the mice was stillstatistically different from that of the control group. Thus, comparedwith FP-05, FP-C1 had a longer-term hypoglycemic effect in the diabeticmouse model. This indicated that the rigid CTP unit in the peptidelinkers could further extend the in vivo functional half-life ofExendin-4.

4.3 Pharmacokinetic Characteristics of Exendin-4-Fc Fusion Proteins inRats

Male SPF SD rats (Shanghai SIPPR-BK Laboratory Animal Co. Ltd.), 4 pergroup, were given a single subcutaneous injection of 0.5 mg/kg FP-C1 orFP-05 after one week of pre-feeding. Blood was taken at 0 h (beforeadministration), 2 h, 8 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, 120 h,144 h (after administration), respectively, about 0.3 mL each time. Theblood collection time points were marked as T₀, T₂, T₈, T₂₄, T₃₂, T₄₈,T₅₆, T₇₂, T₉₆, T₁₂₀, and T₁₄₄, respectively. The blood was allowed tosettle down. Then the serum was separated by centrifugation at 5000 rpmfor 10 min, and the serum samples were stored at −70° C. and analyzed.The concentrations of fusion proteins were determined by thedouble-antibody sandwich ELISA assay. Used for coating was self-made orcommercially available anti-Exendin-4 or GLP-1 N-terminal monoclonalantibody (e.g., Santa Cruz, Cat. No. SC-65389), and used as thedetection antibody was self-made or commercially available horseradishperoxidase-labeled mouse anti-human-IgG-Fc monoclonal antibody (e.g.,Sino Biological Inc., Cat. No. 10702-MM01E-50). The data were input intothe analysis software PKSolver, and pharmacokinetic parameters such asT_(1/2), C_(max) and AUC_((0-t)) of the tested drug were obtained.

As shown in Table 3, the circulation half-life T_(1/2) of 0.5 mg/kg ofFP-05 in rats was 14.9±1.29 h, whereas the T_(1/2) of 0.5 mg/kg of FP-C1in rats was 21.4±2.51 h. The maximum plasma concentration C_(max) ofFP-C1 was significantly higher than that of FP-05. In addition, bycomparing AUC_(0-t) (t=2 h, 5 h, 8 h, 24 h, 28 h, 32 h or 48 h) atdifferent blood collection time points, we could see that at the samedose, the drug exposure of FP-C1 was significantly higher than that ofFP-05, that was, the absolute bioavailability of FP-C1 in rats washigher than that of CTP-free FP-05. It was expected that the clinicaldose of FP-C1 would also decrease.

TABLE 3 Pharmacokinetic parameters of single subcutaneous injection of0.5 mg/kg FP-C1 or FP-C5 in male SD rats T_(1/2) (h) T_(max) (h) C_(max)(ng/mL) AUC(0~∞)ng/mL · h FP-C1 21.4 ± 2.51 24 700.908 35260.92 ±2041.20 FP-C5 14.9 ± 1.29 48 369.167 12535.06 ± 909.42 

4.4 Random Blood Glucose and HbA1c Changes in Db/Db Diabetes Mouse after10 Weeks of FP-C1 treatment.

Female SPF-db/db mice (Shanghai slack experimental animal company), 8weeks old, were fed for 1 week and divided into 4 groups with 6 in eachgroup based on the RBG: model group, Low FP-C1 group (0.75 mg/kg),middle FP-C1 group (1.5 mg/kg), high FP-C1 group (3 mg/kg).Corresponding concentrations of the drug has been hypodermic injected,and PBS has been injected in the model group with 10 ml/kg. Mice in eachgroup were treated with drug once a week for 10 weeks and subjected toblood glucose meter to measure the RBG in each time point. Data wererecorded. Blood sampling points were Od (before drug treatment), 7d,14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d and 70d after drug treatment. Atthe 70^(th) day, mice in each group were blood sampled from the eyesafter a 14h fast and samples were subjected to the HbA1c kit and thecorresponding H700 specific protein analyzer to detect the level ofHbA1c. Results were shown as the percentage of HbA1c in the total bloodproteins.

Date were presented as mean±SD which was generated by SPSS18.0.Differences between the means in the normal distribution were analysedby single factor variance. Homogeneity of variance was examined byDunnett t-test and heterogeneity of variance was examined by Dunnett'sT3 test. Abnormal distribution was examined by non-parametric test.P<0.05 showed significant statically difference.

From the variations of blood glucose of the mice in Table 4, mice inhigh, middle, low FP-C1 groups showed decreased levels of blood glucosecompared with the model group, and their decreased blood glucose levelsshowed dose-dependent. These data indicated that FP-C1 effectively,continuously maintained the blood glucose level in db/db diabetes mice.Moreover, the hypoglycemic effect of the first administration and thelast administration was similar, indicating that no drug resistancereaction occurred, which might lead to the anti-FP-C1 tolerance.

HbA1c was the product that glucose was bound to hemoglobin in the redblood cell, and showed proportional relationship with the glucose levelin the blood. Because the red blood cell has a circulation half-life of120 days, HbA1c represented the total glucose level in the blood in the4-12 weeks before blood sampling, making up the shortcoming that thefasting blood sugar only reflected the transient blood sugar. So, HbA1cwas the most critical indicator of monitoring blood glucose level andalso one of the important factors to be considered in the clinicaltrial. The result of HbAlc in this example could reliably, stablyreflect the blood glucose level 2-3 months before blood sampling. Theamount of HbA1c in the mice with 10 weeks' drug treatment was shown inFIG. 4. The amount of HbA1c in the FP-C1 groups decreased significantlycompared with that of the model group (P<0.01), showing dose-dependent.The amount of HbA1c in the high FP-C1 group decreased remarkably(6.38±1.63) but was still higher than that in the normal C57BL/6J mice(2.5%-3.5%), indicating that 3 mg/kg of FP-C1 would not lead to theoccurrence of long-term hypoglycemia. In conclusion, our results showedthat FP-C1 chronically, effectively, stably controlled the blood sugarlevel in the mice without increasing the risk of long-term hypoglycemia.It was consistent with the trend of blood glucose changes in Table 4.

TABLE 4 The effect of FP-C1 on random blood glucose in db/db mice RBG(mmol/L) Group 7 d 14 d 21 d 28 d 35 d Model 19.5 ± 3.53 23.84 ± 3.28  21.9 ± 4.16 24.08 ± 2.18  21.92 ± 2.39  Low-dose 16.04 ± 1.98   18.5 ±1.38** 17.36 ± 3.87 19.58 ± 3.5*  16.54 ± 3.41  Middle-dose 15.19 ±2.65* 17.34 ± 2.27*  15.42 ± 3.6* 17.82 ± 1.54** 14.02 ± 3.42* High-dose 11.92 ± 1.95** 15.26 ± 3.15**  12.54 ± 3.97** 15.36 ± 3.18** 11.78 ±4.2** RBG (mmol/L) Group 42 d 49 d 56 d 63 d 70 d Model 23.58 ± 3.51 23.66 ± 1.9   21.2 ± 4.72  23 ± 4.09 25.04 ± 5.24  Low-dose 16.02 ±2.07** 19.26 ± 2.17*  13.92 ± 3.53* 16.88 ± 3.92*  17.46 ± 3.0* Middle-dose  14.3 ± 1.49**  16.2 ± 2.86**  12.6 ± 3.53**  14.5 ± 4.37**15.14 ± 2.81** High-dose 12.12 ± 3.95** 15.38 ± 2.43**  9.46 ± 3.45**11.52 ± 3.93** 11.76 ± 5.35** Note: Compared with model group, *P <0.05; **P < 0.01.

4.5 Pharmacodynamic Study of Single Injection of FP-C1 in STZ InducedDiabetes Mice

Male SPF mice (Shanghai slack experimental animal company) with theweight of 25±2 g were divided into the diabetes group and the controlgroup based on their weights. Mice were fed for one week and subjectedto a 18 h fast. The weights of the mice were recorded. The diabetesgroup was treated with 150 mg/kg 1% STZ, pH=4.4 by intraperitonealinjection. While the control group was treated the same volume of citricacid sodium citrate buffer. The RBG of the mice was recorded 10 daysafter the injection. The mice with RBG>16.7h were selected as thediabetes mice. 32 STZ-induced mice were selected and divided into 4groups with 8 in each group to observe the hypoglycemic effect of testdrugs on STZ-induced diabetic mice. 3 mg/kg FP-C1 and 3 mg/kgdulaglutide were injected subcutaneously, respectively. An equal volume(10 mL/kg) of PBS buffer was administered to the diabetes model groupand the control group, respectively. The mice in each group are bloodsampled from tail vein at 0 h (before drug treatment), 1 h, 2 h, 4 h, 6h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h (after drugtreatment). Samples were subjected to the blood sugar meter to measurethe RBG. Data were presented as mean±sd and analyzed by SPSS18.0.Differences between the means in the normal distribution were analysedby single factor variance. The homogeneity of variance was examined byLSD test and the heterogeneity of variance was examined by Dunnett's T3test. Abnormal distribution was examined by non-parametric test. P<0.05indicated significant statistical difference.

FIG. 5 showed the curves of the blood glucose concentration changesduring 0-240 h in STZ-induced mice after single injection of FP-C1,FP-C1 decreased RBG effectively. The blood glucose level in the FP-C1group decreased to the minimum at the 24 h after the FP-C1 treatment.Then it went back slowly but still showed significant differencecompared with that of the model group (P<0.5).

4.6 Study on the Effect of FP-C1 on Weight Loss in Obese Mice Induced byHigh Fat Diet

1. Model Establishment and Drug Treatment

24 C57BL/6J male mice (Shanghai Slack Experimental Animal Company,SCXK(HU): 2012-0002) with 7 weeks old. Feeding environment: temperature22-25° C., relative humidity 45-65%. Lighting time 12h/d. The C57BL/6Jmice were fed for one week and then divided into 3 groups based on theweights: NFD group, HFD group, FP-C1 group (HFD+FP-C1 0.3 mg/kg). TheHFD group and the FP-C1 group were fed with high-fat diet (D12492 highfat diet, Research Dowts CO. USA). The NFD group was fed with normal fatdiet. The FP-C1 group was injected with 0.3 mg/kg FP-C1 every 6 days,while the NFD group and the HFD group were injected with 10 ml/kg PBSbuffer. The mice in each group had a 16 h fast after 96 days and theweights and blood sugar levels were recorded. Blood was sampled andsubjected to 400 g centrifugation for 15 min to obtain the serum. Afterblood was taken, the mice were sacrificed by cervical dislocation, andthe length from nose to anus (body length) was measured to calculate theLee's index. The fat tissue around the epididymis was separated andweighed. The same tissue was stocked in 10% formaldehyde solution forfurther experiment.

2. Detection of Parameters

2.1 Body Weight and Obesity Degree

Mice were weighed every 6 days to draw the curve of body weight and theincreased amount was recorded. Increased amount=body weight at the finalweighing−body weight before grouping. Lee's index was used to describethe obesity degree.

2.2 Fat Mass and its Fraction

Analytical balance was used to weigh the fat tissue around theepididymis. Fat mass fraction was calculated as fat mass fraction=weightof fat tissue (mg)/fasting weight (g).

2.3 Oral Glucose Tolerant Test

After 84 days of experimentation, mice in each group were fasted for 16hours (17:00 am-9:00 pm). The blood glucose meter was used to measurethe fasting blood glucose (FBG). The body weights were recorded. 2 g/kgglucose solution was fed, and the blood sugar levels were detected after30 min, 60 min, 90 min and 120 min of the gavage. The sugar tolerancecurve was drawn and iAUC was calculated based on the modified curve.

2.4 Serum Test

Automatic biochemical analyzer and corresponding kit were used to detectthe concentration of TG and TC in the serum.

2.5 Parameters of Insulin and Islet Tolerance

ELISA was used to detect the concentration of insulin in the serum tocalculate the insulin tolerance.

2.6 Pathological Examination of the Fat Tissue

Fat tissue around the epididymis was subjected to HE dyeing to visualizethe fat tissue cell.

3. Statistics and Analysis

Data were presented as means±SD and analyzed by SPSS18.0. Differencesbetween the means in the Normal distribution were analysed by singlefactor variance. Homogeneity of variance was examined by LSD test andheterogeneity of variance was examined by Dunnett's T3 test. Abnormaldistribution was examined by non-parametric test. P<0.05 showedsignificant statistical difference.

4. Results

4.1 Effect of FP-C1 on Body Weight and Obesity Degree of Mice Fed withHigh-Fat Diet

Compared with those in the NFD group, the body weight, the increasedbody weight and Lee's index all significantly increased (P<0.01). FP-C1effectively decreased the body weight, the increased body weight, andLee's index (P<0.01). Data were shown in Table 5 and FIG. 6.

TABLE 5 The effect of FP-C1 on body weight and Lee's index in high fatdiet induced obese mice Final body Body weight Group weight (g) gain (g)Lee's index NFD 29.14 ± 2.31  5.24 ± 1.09  322.7 ± 11.03  HFD 47.72 ±3.74^(##) 23.9 ± 2.57^(##) 348.78 ± 10.21^(##) FP-C1 33.74 ± 4.5**  9.81 ± 4.24**  327.67 ± 10.28** Note: Compared with normal group, ^(#)P< 0.05, ^(##)P < 0.01; Compared with HFD group, *P < 0.05, **P < 0.01

4.2 Effect of FP-C1 on the Fat Mass and Mass Fraction Around theEpididymis

As shown in Table 6, compared with the NFD group, the mice in HFD groupshowed more fat mass and mass fraction around the epididymis (P<0.01).Compared with the HFD group, mice in FP-C1 group showed decreased fatmass and mass fraction around the epididymis (P<0.05).

TABLE 6 The effect of FP-C1 on Epididymal fat and its index Epididymalfat index Group Epididymal fat (g) (mg/g) NFD 0.68 ± 0.12  25.29 ± 3.83 HFD  2.91 ± 0.45^(##)  64.1 ± 10.85^(##) FP-C1 1.54 ± 0.69* 46.09 ±14.77* Note: Compared with normal group, ^(#)P < 0.05, ^(##)P < 0.01;Compared with HFD group, *P < 0.05, **P < 0.01

4.3 Effect of FP-C1 on the Concentrations of TG and TC in the Serum

Compared with NFD group, the concentrations of TG and TC in the serum inthe HFD group increased significantly (P<0.01). Compared with HFD group,the concentrations of TG and TC in the serum in the FP-C1 groupdecreased significantly (P<0.01). Data shows in Table 7.

TABLE 7 The effect of FP-C1 on serum TG and TC in high fat diet inducedobese mice Group TG (mmol · L⁻¹) TC (mmol · L⁻¹) NFD 1.81 ± 0.31  4.34 ±0.25  HFD 2.93 ± 0.33^(##) 7.77 ± 0.85^(##) FP-C1  1.98 ± 0.38**  5.78 ±0.85** Note: Compared with normal group, ^(#)P < 0.05, ^(##)P < 0.01;Compared with HFD group, *P < 0.05, **P < 0.01

4.4 Effect of FP-C1 on Glucose Tolerance in Mice Fed with High Fat Diet

As shown in FIG. 7, iAUC in the HFD group is significantly higher thanthat in NFD group (P<0.05). Compared with the HFD group, iAUC in theFP-C1 group decreases significantly (P<0.05).

4.5 Effect of FP-C1 on Serum Insulin Concentration and InsulinResistance Index in Mice Fed with High Fat Diet

Compared with those in the NFD group, the insulin concentration and theinsulin resistance index in the HFD group increased significantly, whichmeant that the mice obviously have already obtained insulin tolerance,and they would secreted more insulin to to produce hyperinsulinemia.Compared with those in the HFD group, FP-C1 decreased the INSconcentration (P<0.05) and improved the HOMA-IR (P<0.05) significantly.Results were shown in FIG. 8 and FIG. 9.

4.6 Pathomorphological Examination

HE dyeing showed that the cross-sectional area of fat cells around theepididymis in the HFD group increased significantly compared with theNFD group. Compared with that in the HFD group, the cross-sectional areaof fat cells around the epididymis in the FP-C1 group decreasedsignificantly. Results were shown in FIG. 10.

In conclusion, FP-C1 could control the body weight of the HFD inducedfat mice and has the anti-obesity effect.

Example 5. Production of IL-7-Fc Fusion Proteins and Determination ofBiological Activity and In Vivo Active Half-life

5.1 In Vitro Biological Activity

The stably expressing CHO cell strains of FP-D1 and FP-D2 obtained inExample 1 were cultured in shake flasks under fed batch conditions for12-14 days. The fusion proteins were purified by Protein A affinitychromatography. The purity of both the fusion proteins was above 95%,and the molecular sizes were also as expected. The fusion proteins werethen used for activity analysis. The in vitro biological activityanalysis of IL-7 and its fusion proteins was performed as follows. Themouse spleen-derived mononuclear cells were activated by concanavalin A(ConA), and then 100 μL of the cells were seeded into each well of a96-well plate, followed by addition of a series of concentrationgradient of FP-D1 or FP-D2. The cells were cultured at 37° C., 5% CO₂for 72 h, and after addition of 20 μL of MTT reagent, were continued toculture for 4 h. The medium was aspirated and 100 μL of dimethylsulfoxide (DMSO) was added to each well. The absorbance at 492 nm wasused to determine the proliferation of the cells. Three replicates wereperformed for each concentration gradient, and each replicate wasmeasured twice. Recombinant IL-7 (hIL-7, Sino Biological Inc.) was usedas positive control and the medium was used as negative control. FIG. 11showed the ability of hIL-7, FP-D1 and FP-D2 fusion proteins tostimulate the proliferation of mouse mononuclear cells. Based on thedose response curves plotted, the half maximal effective concentrationvalues (EC₅₀) of the FP-D1 and FP-D2 fusion proteins were 0.039 and0.048 nM, respectively. The recombinant fusion proteins exhibited betterin vitro bioactivity than hIL-7 that E. coli expressed (EC₅₀ value of0.08 nM in terms of molarity).

5.2 Determination of Pharmacokinetic Parameters of IL-7-Fc FusionProteins

Male SPF SD rats (Shanghai SIPPR-BK Laboratory Animal Co. Ltd.), 3 pergroup, were given a single intravenous (iv) or subcutaneous (sc)injection of 2 mg/kg FP-D1 or FP-D2 after one week of pre-feeding. Thechanges of blood drug concentration with time were investigated. At 0 h,3 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 126 h, 144 h, and 172 h afterinjection, blood was collected, about 0.3 mL each time. The blood wasallowed to settle down. Then the serum was separated by centrifugationat 5000 rpm for 10 min and taken for assay. The fusion proteinconcentration at each time point was determined by an ELISA methodspecific for human IL-7. The data were analyzed by the PKSolversoftware, and pharmacokinetic parameters such as T_(1/2) and AUC_((0-t))were calculated. The results were shown in Table 8.

TABLE 8 Pharmacokinetic parameters of fusion proteins AUC 0-inf_obsVz/F_obs Cl/F_obs T_(1/2)(h) (ng/mL*h) ((μg)/(ng/mL)) ((μg)/(ng/mL)/h)Drug iv sc iv sc iv sc iv sc FP-D1 18.760 ± 19.870 ± 24458.54 ± 10310.28± 0.526 ± 1.293 ± 0.020 ± 0.045 ± 1.109 2.287 8739.60 1364.23 0.1430.173 0.006 0.005 FP-D2 12.86 ± 13.81 ± 12025.16 ± 4497.43 ± 0.79 ± 0.11± 0.04 ± 2.20 ± 4.32 4.47 2074.52 571.93 0.32 0.01 0.01 0.75

The data showed that the elimination half-lives of FP-D2 were 12.86 h(iv) and 13.81 h (sc), respectively, which fit with linear metabolickinetics. The elimination half-lives of FP-D1 were 18 h (iv) and 19 h(sc), respectively, slightly longer than those of FP-D2. The overallclearance rate (C1) of FP-D1 by intravenous administration was less thanthat of FP-D2, indicating that after FP-D2 was modified to generateFP-D2, the in vivo clearance rate of FP-D1 was slowed down compared withthat of FP-D2. In addition, we also found that due to the introductionof the CTP structure, the apparent volume of distribution of FP-D1 wasreduced. This indicated that FP-D1 was less distributed in tissues andhad a higher concentration in the blood, thus having a higher in vivoexposure. FP-D1 had the rigid CTP structure compared with FP-D2, whichreduced the clearance, reduced the apparent volume of distribution, andincreased AUC_((0-t)). Therefore, the bioavailability of FP-D1 washigher, and the efficacy of FP-D1 might be better than that of FP-D2. Itwas expected that the clinical dose of FP-D1 would also decrease. Thus,FP-D1 exhibited excellent performance in terms of biological activityand pharmacokinetics. The results of this experiment indicated that thehighly sialylated, negatively charged CTP in the peptide linkers couldresist the clearance of the kidney, further prolong the half-lives ofthe fusion proteins, and increase the bioavailability of the fusionproteins.

Example 6. Production of hGH-Fc Fusion Proteins and Determination ofBiological Activity and In Vivo Half-life

6.1 Determination of In Vitro Biological Activity of Fusion Proteins byMTT Assay

The stably expressing CHO cell strains of FP-E1 and FP-E2 obtained inExample 1 were cultured in shake flasks under fed-batch conditions for12-14 days. The fusion proteins were purified by Protein A affinitychromatography. The purity of the fusion proteins was above 95%, and themolecular sizes were as expected. The fusion proteins were then used foractivity analysis. In vitro hGH biological activity could be determinedby measuring proliferation of Nb2 rat lymphoma cells. Since the Nb2cells responded to the stimulation of hGH on the lactating receptors ofthe Nb2 cells to proliferate, the Nb2 cell proliferation assay could beused to evaluate the biological activity of growth hormone (Uchida H etal., 1999, J Mol Endocrinol, 23: 347-353).

Nb2-11 rat lymphoma cells (ATCC) were cultured in Fischer's mediumcontaining 10% FBS. The fusion protein was diluted with serum-freemedium, diluted from 1000 ng/mL to 8 gradients at 1:3 ratio, and addedto a 96-well plate, 100 μL per well, with the last column using themedium as negative control. The cells growing in the log phase werewashed with serum-free medium, and adjusted to a density of 3×10⁶cells/mL. 100 μL cell suspension was added to each well of the above96-well plate. The cells were continued to culture for 48 h in a 37° C.,5% CO₂ incubator, and cell proliferation was measured using a CCK-8 kit(Cell Counting Kit, Cat. No. 40203ES80, Shanghai Yisheng BiotechnologyCo., Ltd.). The absorbance at 450 nm was measured using a microplatereader and the OD readings were plotted against the concentrations ofthe fusion protein. The biological activity of the fusion protein couldbe determined from the resulting dose response curve.

FIG. 12 showed the ability of hGH fusion proteins to stimulate theproliferation of Nb2 cells. Table 9 showed the EC₅₀ values for differentfusion proteins. Since the amino acids at the C-terminus of growthhormone were closely related to its function, direct linking of Fc tothe C-terminus of hGH might affect its biological activity. As thepeptide linker was added between hGH and Fc, the activity of the hGHfusion protein was increased. As can be seen from the results, theactivity of FP-E1 was nearly doubled compared with that of FP-E2. Thismight be due to the fact that CTP peptide did not only play its role inprolonging the half-life of the fusion protein, but also the rigidstructure of CTP peptide together with a flexible peptide acted as alinker connecting Fc to the target protein. This novel peptide linkerfacilitated folding of the fusion protein into a stablethree-dimensional structure, thus increasing the biological activity ofhGH.

TABLE 9 EC₅₀ values of hGH fusion proteins Fusion protein hGH FP-E1FP-E2 EC₅₀ (pM) 6.94 17.3 27.84

6.2 In Vivo Circulation Half-Life

Male SPF SD rats (Shanghai SIPPR-BK Laboratory Animal Co. Ltd.),weighing about 290 g, were divided into groups after one week ofpre-feeding, with 3 in each group, and were given a single intravenousinjection of 0.176 mg/kg FP-E1 or FP-E2. The changes of blood drugconcentration with time were investigated. For the control group and thedrug-administered group, blood was collected at 0 h, 0.5 h, 1 h, 2 h, 3h, 4 h, 5 h, 8 h, 10 h, 24 h, 48 h, and 72 h after administration. Theblood was placed at room temperature for 30 min and then centrifuged at5000 rpm for 10 min. The serum was separated and stored at −20° C. SerumhGH levels at various time points were determined using an ELISA assayspecific for hGH. The main pharmacokinetic parameters of each group werecalculated by the PKSolver software. The pharmacokinetic parameters ofeach group were shown in Table 10.

TABLE 10 Pharmacokinetic parameters of hGH fusion proteins Tmax CmaxMRT(0-∞) Vz(μg)/ Cl(μg)/ Drug T_(1/2)(h) (h) (ng/mL) AUC_((0~t)) (h)(ng/mL) (ng/mL)/h FP-E1 2.39 ± 0.5 h 4259.4396 16483.295 ± 3.34 ± 0.027± 0.008 ± 0.37 4483.39 0.43 0.002 0.002 FP-E2 2.57 ± 0.5 h 2414.74998987.14 ± 3.33 ± 0.054 ± 0.015 ± 0.04 986.33 0.01 0.008 0.002

From the results, the in vivo half-lives of FP-E1 and FP-E2 fusionproteins were 2.6 h and 2.4 h, respectively. The half-lives werebasically the same, and this could also be seen from the MRT parameters.The times that FP-E1 and FP-E2 stayed in the body were also close.According to the blood drug concentration-time curve of FIG. 13, theblood concentration of FP-E1 was always higher than that of FP-E2. Itwas speculated that structural changes of FP-E1 resulted in the changesin the properties of its pharmacokinetics. The total body clearance ofFP-E1 was only half of that of FP-E2, indicating that the in vivoclearance of FP-E1 was relatively slow. The apparent volume ofdistribution of FP-E2 was twice that of FP-E1, indicating that FP-E2 wasrapidly distributed into tissues after entering the body, causing itsblood concentration to be lower than that of FP-E1. FP-E1 had the rigidCTP structure compared with FP-E2, which reduced the clearance,increased AUC_((0-t)), and reduced the apparent volume of distribution.This indicated that FP-E1 was less distributed in tissues and had higherconcentration in the blood, resulting in higher in vivo exposure. So thestructural advantage of FP-E1 was reflected not only in the superiorpharmacokinetic parameters, but also in that its efficacy could bebetter than that of FP-E2. FP-E1 had a higher blood concentration due tothe structural modifications, which meant that its bioavailability washigher. So FP-E1 was better than FP-E2, and its clinical dose could beexpected to decrease. Thus, FP-E1 exhibited superior performance interms of biological activity and pharmacokinetics.

6.3 Determination of In Vivo Biological Activity of Fusion Proteins

Male Sprague-Dawley rats (SPF grade with four weeks age and 60-80 g bodyweight) were provided by the Experimental Animal Center of ChinaNational Institutes for Food and Drug Control.

The pituitary gland was removed by surgery with two week's recoveryperiod. Before administration, the qualified healthy animals wereselected, whose body weight changes were less than ±10% of thepreoperative weights. The pituitary gland-removed rats were randomlydivided into 6 groups, 5 animals per group. Regular rhGH (trade namenorditropin, Novo Nordisk A/S, activity of 3 IU/mg) was used as thepositive reference drug 1 (γ1) in this experiment. PEG-rhGH (ChangchunGeneScience Pharmaceuticals Co., Ltd., activity of 6 IU/mg) was used asthe positive reference drug 2 (Y2). Low, median and high doses of FP-E1were set at 5 mg/kg/14d, 15 mg/kg/14d, and 45 mg/kg/14d, respectively.Based on the molecular weight and the number of molecules, the mediandose of FP-E1, 15 mg/kg/14d, was comparable to the positive referencedrugs Y1 and Y2. The mode of administration was subcutaneous injectionin the neck. Different doses of FP-E1 fusion protein and Y2 wereadministered to each rat once on the first day and the other on theeighth day. Y1 was administered once a day for 14 consecutive days.

Each rat was weighed daily after administration. All rats weresacrificed by carbon dioxide asphyxiation on the 15th day, and weighed.The difference between the day i (di) body weight (bw_(i)) of eachanimal and the body weight before administration, i.e. the day 1 (d1)body weight (bw₁), was the increased body weight. If necessary, anautopsy could be performed at the end of the experiment. The sellaregion was cut, and the presence or absence of pituitary residue wasexamined by the naked eye. The animals with residual pituitary glandwere removed. The weight gain calculation formula was:Δbw=bw_(i)−bw_(i), wherein Δbw was the weight gain; bw₁ was the d1 bodyweight before administration; and bw_(i) was the day i body weight afteradministration. The measurement data were expressed as mean±standarddeviation (M±SD), and the results were shown in Table 11. FIG. 14 showedthe growth curves of all groups after administration.

TABLE 11 Body weight changes of rats before and after hGH fusion proteinadministration (M ± SD, n = 7) Total dose bw (g) before Δbw (g) afterfor 2 administration administration Group weeks mg/kg d 1 d 8 d 15Control 0 72.4 ± 6.9  0.7 ± 1.4  1.3 ± 1.7 Y1 5.8 73.1 ± 5.2 15.1 ± 2.523.3 ± 4.4 Y2 2.9 72.2 ± 6.4 15.3 ± 2.3 25.3 ± 4.3 Low dose of 5 74.3 ±6.8 10.5 ± 1.3 22.4 ± 2.9 FP-E1 Median dose of 15 72.4 ± 4.8 13.7 ± 3.127.3 ± 6.8 FP-E1 High dose of 45 73.6 ± 5.9 17.3 ± 2.1 37.9 ± 3.0 FP-E1

As could be seen from the results, compared with the control group, eachof the administration groups had its body weight significantly increasedon the 8th day after administration, but the differences of Δbw betweenthe administration groups were not significant. On the 15th day afteradministration, FP-E1 had a very significant promotive effect on thebody weight gain of rats. The weight gain (Δbw) of pituitary-removedrats induced by the high dose FP-E1 was about 1.5 times those of therhGH (Y1) and PEG-rhGH (Y2) groups. The weight gain induced by themedian dose FP-E1 was slightly larger than that of the Y1 group, and wasbasically equal to that of the Y2 group. We found that the high doseFP-E1 group had their body weights significantly increased after thesecond administration, and FP-E1 showed a more significant promotiveeffect on weight gain than PEG-rhGH. However, within one week after thefirst administration, the Δbw values of the high dose FP-E1 and PEG-rhGHgroups were not statistically different. Based on the analysis of theΔbw differences, it was deduced that FP-E1 should have a longer in vivoactive half-life than PEG-rhGH. Thus, after repeated administration,this in vivo drug accumulation effect caused the upward trend of thegrowth curve of the FP-E1 group steeper after the second administration.

Example 7. Production and Biological Activity Analysis of Anti-CD3×CD20Bispecific Antibodies

7.1 Preparation and Identification of Anti-CD3×CD20 BispecificAntibodies

The stably expressing CHO cell strains of FP-F1 and FP-F2 obtained inExample 1 were cultured in shake flasks under fed-batch conditions for10-14 days. The bispecific antibodies were purified by Protein Aaffinity chromatography. The purity of both the fusion proteins was morethan 95%, and the molecular sizes were as expected. The proteins werethen used for activity analysis.

7.2 Determination of In Vitro Activity of Anti-CD3×CD20 BispecificAntibodies

PBMCs were prepared from fresh human blood by density gradientcentrifugation and resuspended at 5×10⁶ cells/mL with medium containing10% heat inactivated FBS for later use. The antibody (anti-CD3monoclonal antibody OKT3 or bispecific antibody FP-F1) was diluted to 2μg/mL using complete medium, and then diluted to 8 gradients at 1:5ratio. The diluted antibody was added to a 96-well plate at 100 μL perwell in triplicate. The medium was used as negative control and ConA wasused as positive control. The PBMC cell suspension prepared as above wasadded to the 96-well plate at 100 μL/well, and cultured at 37° C., 5%CO₂ for 72 h. After incubation, the culture supernatant in the 96-wellplate, 100 μL per well, was carefully aspirated, and the IFN-γ contentin the supernatant was measured by an IFN-γ ELISA kit (BD Biosciences)(FIG. 15). 10 μL of the CCK-8 reagent was added to each well of the96-well plate, and the incubation was continued for 4 h. Using theantibody concentration as the X-axis and the absorbance value at 492 nmas the Y-axis, a four-parameter S-curve was fitted and the EC₅₀ valuewas calculated (FIG. 16). The results showed that the amount of IFN-γproduced by FP-F1-activated T cells was positively correlated with theantibody concentration and slightly lower than that produced by thecontrol antibody OKT3. This indicated that the anti-CD3 single chainantibody of the bispecific antibody functioned well. The EC₅₀ value thatOKT3 activated human PBMCs was 0.015 nM, whereas that of FP-F1 was 0.233nM. The activation ability of FP-F1 decreased by about two orders ofmagnitude, which helped to reduce the clinical toxicity of thebispecific antibody.

Another anti-CD3×CD20 bispecific antibody FP-F2 constructed in thepresent invention also activated T cells and produced IFN-γ in aconcentration-dependent manner in the human PBMC cell activation assay.The EC₅₀ that FP-F2 activated human PBMCs was 0.214 nM, which wascomparable to that of FP-F1.

7.3 Efficacy of Anti-CD3×CD20 Bispecific Antibody FP-F1 in KillingSubcutaneous Xenografts

Human Burkitt's lymphoma Raji Cells (8×10⁶ cells, The Collection CellBank of Chinese Academy of Sciences) and Matrigel (BD Biosciences, Cat.No. 354234) were co-inoculated in female SCID Beige mice (ShanghaiLingchang Biotechnology Co., Ltd.) at a ratio of 1:1 subcutaneously.After 6 days of growth, the mice were grouped according to body weightand tumor volume, 7 mice per group. The cultured LAK cells were injectedinto the tumor tissue at a dose of 1×10⁶ cells/50 μL. Meantime, FP-F1was administered in the same day at doses of 10, 1 and 0.1 mg/kg,respectively, twice a week by intravenous injection. The control groupswere: (1) negative control group: solvent of FP-F1 (PBS); (2) positivecontrol group: Rituxan® (anti-CD20 antibody, Genentech), administered ata dose of 10 mg/kg, twice a week by intravenous injection. The bodyweight and tumor volume of the mice were measured twice a week. Thevolume was calculated with ½×length×width×width (mm³).

In FIG. 17, different doses of FP-F1 showed good tumorgrowth-inhibition. The tumors in 2 mice in the 10 mg/kg group completelydisappeared. The tumor in 1 mouse in the 1 mg/kg group had completelydisappeared. The 0.1 mg/kg dose administered to one group also showed acertain effect of inhibiting tumor growth. The tumor in the negativecontrol group grew normally and reached 400 mm³ on the 14th day. Theresults showed that the therapeutic effect of the 1 mg/kg FP-F1 dose wasequivalent to that of the 10 mg/kg Rituxan dose, indicating that theeffective dose of the bispecific antibody FP-F1 constructed in thepresent invention for inhibiting tumor growth in vivo was only 1/10 ofthat of the anti-CD20 monoclonal antibody (Rituxan®). It was expectedthat the clinical dose of FP-F1 would be greatly reduced.

All documents mentioned in the present invention are hereby incorporatedby reference to the same extent as if each of the documents isindividually recited for reference. It is to be understood that variouschanges and modifications may be made by those skilled in the art uponreading the above teachings of the present invention, which also fallwithin the scope of the claims appended hereto.

What is claimed is:
 1. A fusion protein having the structural formula ofK1-L-K2 or K2-L-K1, wherein K1 is a first biologically active molecule;L is a linker peptide between K1 and K2, which comprises a flexiblepeptide and a rigid peptide, wherein the flexible peptide comprises oneor more flexible units and the rigid peptide comprises one or more rigidunits, wherein the flexible unit comprises an amino acid sequenceselected from the group consisting of: i) (SEQ ID NO: 21)GSGGGSGGGGSGGGGS; ii) (SEQ ID NO: 22) GSGGGGSGGGGSGGGGSGGGGSGGGGS; iii)(SEQ ID NO: 23) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; iv)(SEQ ID NO: 24) GSGGGGSGGGGSGGGGS; v) (SEQ ID NO: 25)GGGSGGGSGGGSGGGSGGGS; and vi) (SEQ ID NO: 26) GGSGGSGGSGGS;

wherein the rigid unit comprises an amino acid sequence selected fromthe group consisting of: i) SSSSKAPPPSLPSPSRLPGPSDTRILPQ;(SEQ ID NO: 27) ii) (SEQ ID NO: 28) PRFQDSSSSKAPPPSLPSPSRLPGPSDTRILPQ;iii) (SEQ ID NO: 29) SSSSKAPPPS; iv) (SEQ ID NO: 30) SRLPGPSDTRILPQ; andv) (SEQ ID NO: 31) SSSSKAPPPSLPSPSR;

and wherein K2 is a second biologically active molecule, and wherein K1or K2 each is selected from the group consisting of a protein, a proteindomain, a peptide, and an antibody or an antigen-binding fragmentthereof.
 2. The fusion protein of claim 1, wherein K1 is a soluble ormembrane signal molecule, a cytokine, a growth factor, a hormone, acostimulatory molecule, an enzyme, a receptor, or a protein or peptidewhich is a ligand to a receptor, and wherein K2 is a protein or aprotein domain which prolongs the circulation half-life of K1.
 3. Thefusion protein of claim 2, wherein K2 is selected from the groupconsisting of a human hemoglobin, an iron Transferrin, and an Fcfragment of an immunoglobulin.
 4. The fusion protein of claim 1, whereinK1 is selected from the group consisting of a toxin, an enzyme, acytokine, a membrane protein, and an immunoregulatory cytokine; K2 is anantibody or an antigen-binding fragment thereof; and K1 is connected toK2 via L to form an antibody fusion protein.
 5. The fusion protein ofclaim 1, wherein K1 is selected from the group consisting of anadenosine A1 receptor, angiotensin-converting enzyme (ACE), Activinfamily, ADAM family, ALK family, α-1-antitrypsin, programmed cell deathprotein family, nerve growth factor and receptor family, bonemorphogenetic protein (BMP) and receptor family, complement factor,calcitonin, cancer associated antigen, cathepsin family, CCL chemokineand receptor family, CD superfamily, CFTR, CXCL chemokine and receptorfamily, EGF and receptor family, blood coagulation factor IIa, factorVII, VIII, IX, ferritin, fibroblast growth factor (FGF) and receptorfamily, follicle stimulating hormone, FZD family, HGF, glucagon, Cardiacmyosin, growth hormone, Ig, IgA receptor, IgE, insulin-like growthfactor (IGF) and binding protein family, interleukin (IL) and receptorsuperfamily, interferon (INF) family, iNOS, integrin family, kallikreinprotein family, laminin, L-selectin, luteinizing hormone, MMP family,mucin-like family, cadherin superfamily, platelet-derived growth factor(PDGF) and receptor family, parathyroid hormone, serum albumin, T-cellreceptor superfamily, TGF-α, transforming growth factor-β superfamily,thyroid stimulating hormone, parathyroid stimulating hormone, tumornecrosis factor (TNF) superfamily and receptor TNFRSF superfamily,urokinase, WNT signaling pathway family, thymosin α1, thymosin β4, andVEGF and receptor family.
 6. A fusion protein having the structuralformula of K1-L-K2 or K2-L-K1, wherein K1 is human coagulation factorVII, human coagulation factor VIII, Exendin-4, human interleukin 7, orhuman growth hormone; L has an amino acid sequence of any one of SEQ IDNOs: 2-7; and K2 is an Fc fragment of a human IgG, IgM, or IgA or avariant thereof, and the Fc variant comprises at least one amino acidmodification in the wild-type human IgG Fc domain and has reducedeffector functions and/or an enhanced binding affinity for a neonatalreceptor FcRn.
 7. The fusion protein of claim 6, wherein K2 compriseshuman immune protein Fc or a variant thereof.
 8. The fusion protein ofclaim 6, wherein K2 comprises an amino acid sequence of any one of SEQID NOs: 8-12.
 9. The fusion protein of claim 6, wherein K1 is Exendin-4having the amino acid sequence of SEQ ID NO:
 15. 10. A method for thetreatment or prophylaxis of type II (non-insulin dependent) diabetes,comprising administering an effective amount of the fusion protein ofclaim 9 to a subject suffering from type II (non-insulin dependent)diabetes.
 11. A method for the treatment or prophylaxis of obesities,comprising administering an effective amount of the fusion protein ofclaim 9 to a subject suffering from obesities.
 12. The fusion protein ofclaim 6, wherein K1 is human coagulation factor VII (FVII) having theamino acid sequence of SEQ ID NO: 13, human coagulation factor VIII(FVIII) having the amino acid sequence of SEQ ID NO: 14, interleukinIL-7 having the amino acid sequence of SEQ ID NO: 16, or human growthfactor having the amino acid sequence of SEQ ID NO:
 17. 13. A fusionprotein having the structural formula of K1-L-K2 or K2-L-K1, wherein K1comprises one antibody or an antigen-binding fragment thereof; K2comprises another antibody or an antigen-binding fragment thereof; andK1 is connected to K2 by L to form a bispecific antibody, wherein thelinker peptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-7.
 14. The fusion protein of claim 13,wherein K1 is a double chain antibody of anti-CD20, and K2 is a singlechain antibody of anti-CD3.
 15. The fusion protein of claim 14, whereinthe double chain antibody of anti-CD20 comprises a heavy chain havingthe amino acid sequence of SEQ ID NO: 18 and a light chain having theamino acid sequence of SEQ ID NO: 19; the single chain antibody ofanti-CD3 comprises the amino acid sequence of SEQ ID NO: 20; and Lcomprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:
 6. 16. Amethod of preparing or producing the fusion protein of claim 1,comprising a step of linking K1 and K2 by L.
 17. The method of claim 16,comprising the following steps: (a) ligate DNA sequences encoding K1 andK2 by a DNA sequence encoding L to form a fusion gene, (b) introduce thefusion gene obtained in step (a) into a eukaryotic or prokaryoticexpression host, (c) culture by fermentation a high yield expressionstrain obtained in step (b) through screening to express the fusionprotein, and (d) harvest the fermentation broth from step (c) andisolate and purify the fusion protein.
 18. The fusion protein of claim1, wherein the rigid peptide comprises 1, 2, 3, 4, or 5 rigid units. 19.The fusion protein of claim 1, wherein the linker peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:2-7.